In recent decades Plasmodium falciparum has re-emerged as the major health threat in most tropical regions of the world. We have recently demonstrated, in vitro, that 5-fluoroorotate is exceedingly toxic to P. falciparum (IC30=6x10-9M). In contrast, 5-fluoroorotate is much less toxic to human fibrosarcoma cells (IC30=5x10-5M). Furthermore, the combination of uridine and 5-fluoroorotate says mammalian cells from toxicity without compromising the antimalarial activity of 5-fluoroorotate. Finally, 5- fluoroorotate is very effective against multiple drug-resistant parasites. The primary goal of the proposed research is to determine the molecular basis for the selective antimalarial activity of 5-fluoroorotate. We will characterize the transport of 5-fluoroorotate into erythrocytes infected with asynchronous as well as synchronous populations of P. falciparum. We will also follow the metabolism of radioactive drug into nucleotides and into macromolecules such as RNA, and the enzyme thymidylate synthetase. Experiments will be performed to determine whether inactivation of malarial thymidylate synthetase plays a role in whether inactivation of malarial thymidylate synthetase plays a role in whether inactivation of malarial thymidylate synthetase plays a role in 5-fluoroorotate toxicity. Kinetic interactions between parasite 5-fluoro 2' -deoxyuridylate will be analyzed. A new antimetabolite, trans 5-fluorodihydrocrotate will be synthesized and tested as a selective antimalarial drug. If successful in vitro, it will tell us that derivatives of dihydroorotate may also be highly effective antimalarial drugs.